This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We wish to carry out quantitative real-time imaging of the kinase ERK in synthetic mammalian MAP kinase cascade that we have constructed in saccharomyces cerevisiae. Using novel genetically encoded FRET sensors that are specific for ERK, we propose to use 2-photon fluorescence lifetime imaging to quantify its activation dynamics, both temporarily and spatially. The two FRET sensors that we have are based on the fluorescent pairs EGFP-mRFP1 and Cerulean-Venus. We intend to follow the method that was published recently by the group that developed the sensors.